Selective estrogen receptor modulator: A novel polysaccharide from Sparganii Rhizoma induces apoptosis in breast cancer cells.

A polysaccharide named SpaTA, as novel selective estrogen receptor modulator, was isolated from water extraction of traditional Chinese herbal medicine Sparganii Rhizoma. SpaTA had a backbone consisting of 2-O-grailsine-ß-xylose (4→6)-α-glucose (1→4) -ß-mannose osamine. There is an aluminium element combined with nitrogen on both grailsine and mannose osamine in repeating unit of SpaTA. The anticancer effect of SpaTA was assessed using ZR-75-1 human breast cancer cells. The results showed that SpaTA induced sequential increases in proliferation and apoptosis through a time- and concentration-dependent manner. Further studies revealed that SpaTA regulated the expression and nuclear translocation of ERα, then modulated the downstream estrogen signaling pathway. Moreover, knock-down ERα in ZR-75-1 cells and overexpress ERα in MDA-MB-231 cells also provided evidences that SpaTA activated the apoptosis-related caspase -3, -8, -9 and PARP in an ERα-dependent manner. Taken together, these results indicated that SpaTA can induce the apoptosis of breast cancer cells through regulating ERα. Therefore, SpaTA may be considered as an effective agent against human breast cancer.

Perfluorooctane sulfonate-induced testicular toxicity and differential testicular expression of estrogen receptor in male mice.

Perfluorooctane sulfonate (PFOS, CAS#1763-23-1) causes male reproductive toxicities, but the underlying mechanisms are still unclear. In this study, 0, 0.5 and 10mg/kg/day PFOS were given by oral gavage to adult mice for 5 weeks. In the 10mg/kg group, serum testosterone levels decreased significantly. Sperm counts declined which might be associated with the decreased proliferation and increased apoptosis of germ cells. In relation to increased apoptosis, bax, cleaved caspase-9 and cleaved caspase-3 levels elevated significantly, indicating that PFOS induced germ cell apoptosis by activating the mitochondrial pathway. In addition, the increase in levels of testicular estrogen receptor (ER) ß was observed in both 0.5 and 10mg/kg group, whereas a decrease in ERα expression was only observed in 10mg/kg group. These results suggested that the alterations in testicular ERs expression, together with decreased proliferation and increased apoptosis of germ cells, might be involved in PFOS-induced testicular toxicity.

Effects of di-n-butyl phthalate on male rat reproduction following pubertal exposure.

Di-n-butyl phthalate (DBP) is an endocrine-disrupting chemical that has the potential to affect male reproduction. However, the reproductive effects of low-dose DBP are still not well known, especially at the molecular level. In the present study, pubertal male Sprague-Dawley rats were orally administered DBP at a wide range of doses (0.1, 1.0, 10, 100 and 500 mg kg⁻¹ day⁻¹) for 30 days. The selected end points included reproductive organ weights, testicular histopathology and serum hormonal levels. Additionally, proteomic analysis was performed to identify proteins that are differentially expressed as a result of exposure to DBP at low doses (0.1, 1.0 and 10 mg kg⁻¹ day⁻¹). Toxic effects were observed in the high-dose groups, including anomalous development of testes and epididymides, severe atrophy of seminiferous tubules, loss of spermatogenesis and abnormal levels of serum hormones. Treatment with low doses of DBP seemed to exert a 'stimulative effect' on the serum hormones. Proteomics analysis of rat testes showed 20 differentially expressed proteins. Among these proteins, alterations in the expression of HnRNPA2/B1, vimentin and superoxide dismutase 1 (SOD1) were further confirmed by Western blot and immunohistochemistry. Taken together, we conclude that high doses of DBP led to testicular toxicity, and low doses of DBP led to changes in the expression of proteins involved in spermatogenesis as well as changes in the number and function of Sertoli and Leydig cells, although no obvious morphological changes appeared. The identification of these differentially expressed proteins provides important information about the mechanisms underlying the effects of DBP on male rat reproduction.

[Effects of di-butyl phthalate on the reproductive system of adolescent male rats].

OBJECTIVE: To explore the effects of di-butyl phthalate (DBP) on the reproductive system of adolescent male rats. METHODS: Sprague-Dawley (SD) rats aged 5 weeks were assigned to receive corn oil (vehicle control) or DBP orally at 10, 100 and 500 mg/(kg x d) for 30 days. After the exposure, the testis, epididymis, liver and pituitary of the rats were weighted and their ratios to the body weight obtained. Histopathological changes of the testis and epididymis were examined by Hematoxylin-eosin staining, the levels of testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the serum were measured by radioimmunoassay, and the relative mRNA expressions of the steroidogenesis acute regulatory protein (StAR), proliferating cell nuclear antigen (PCNA), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and scavenger receptor (SR) were detected by real-time quantitative RT-PCR. RESULTS: DBP induced significant histopathological changes in the testicular tissue at 100 and 500 mg/(kg x d), and decreased the testicular and epididymal weights, inhibited the mRNA expressions of StAR and PCNA, reduced the levels of T and LH, and elevated the level of FSH at 500 mg/(kg x d). At the dose of 10 mg/(kg x d), DBP increased serum LH and FSH and the mRNA expression of P450scc. While the SR mRNA expression showed no significant changes in all the groups. CONCLUSION: High level of DBP has apparent toxic effect on reproductive system of male rats. Low - dose DBP can increase the level of serum gonadotropin LH and affect the mRNA expression of P450scc in the testis.

Effects of permethrin, cypermethrin and 3-phenoxybenzoic acid on rat sperm motility in vitro evaluated with computer-assisted sperm analysis.

Pyrethroid pesticides, produced and used worldwide, have been reported to impair male reproductive function by reducing sperm count and sperm motility. They are divided into two types: type I pyrethroids including permethrin, etc. and type II pyrethroids including cypermethrin, fenvalerate, cyfluthrin, etc. Our previous study showed that fenvalerate and cypermethrin could reduce sperm motility in vitro. However, it is not clear whether permethrin and 3-phenoxybenzoic acid (3-PBA, the major metabolite of pyrethroids) affect sperm motility directly or indirectly by affecting spermatogenesis via interaction with androgens and/or their receptors. In this study, rat sperm suspensions were treated respectively with permethrin, cypermethrin and 3-PBA, at various concentrations (0, 1, 4, 16, or 64mmol/L) for various times (1, 2, or 4h). The motility parameters of sperm were analyzed with a computer-assisted sperm analysis (CASA) system. The differential effects of permethrin and cypermethrin on sperm motility patterns in vitro were also compared. Our study revealed that permethrin and cypermethrin could reduce sperm motility in vitro in a concentration- and time-dependent manner. Marked differences between the two pyrethroids were not found in this study. Moreover, 3-PBA did not reduce sperm motility directly at all concentrations and treatment periods. These results provide further evidence that permethrin and cypermethrin can directly affect mature rat sperm motility.

Effects of fenvalerate and cypermethrin on rat sperm motility patterns in vitro as measured by computer-assisted sperm analysis.

Fenvalerate and cypermethrin were reported to impair male reproductive function, inducing significant reductions in epididymal sperm count. Further, fenvalerate was shown to reduce sperm motility. However, it is not clear whether fenvalerate and cypermethrin might impact sperm motility directly or indirectly by affecting spermatogenesis via interaction with androgens or their receptors. In this study, sperm suspensions were treated with fenvalerate and cypermethrin, respectively, at various concentrations (0, 1, 4, 16, or 64 micromol/L) for various times (1, 2, or 4 h). The motility parameters of sperm treated with these two insecticides were analyzed with a computer-assisted sperm analysis (CASA) system. The differential effects of fenvalerate and cypermethrin on rat sperm motility patterns in vitro were also compared. Our study revealed that fenvalerate and cypermethrin reduced sperm motility in vitro in a concentration- and time-dependent manner. Cypermethrin exerted a greater effect on sperm motility in comparison to fenvalerate. These results provided evidence that fenvalerate and cypermethrin directly influence mature rat sperm motility.

Fenvalerate inhibits progesterone production through cAMP-dependent signal pathway.

Fenvalerate is a widely used synthetic pyrethroid insecticide and is known to impede the male reproductive function. However, the mechanisms remain to be elucidated. In this study, mouse Leydig tumor cells (MLTC-1) were used to investigate the effects of fenvalerate on progesterone production. Fenvalerate treatment inhibited progesterone secretion induced by human chorionic gonadotropin (hCG), cholera toxin (CT) or forskolin and decreased cAMP levels induced by hCG, but not by CT or forskolin, which suggested a repaired site on the upstream components of G protein or G protein per se by fenvalerate in the cAMP-mediated signal pathway. Furthermore, the addition of cAMP analog, 8-Br-cAMP, could not reverse fenvalerate-suppressed progesterone synthesis, indicating that fenvalerate interfered with the downstream molecules of cAMP. In addition, fenvalerate decreased steroidogenic acute regulatory protein (StAR) mRNA and protein levels, and also profoundly inhibited the activity of P450 side chain cleavage enzyme (P450scc) which was consistent with the decreased expression of P450scc mRNA and protein in MLTC-1 cells. These results suggested that fenvalerate might inhibit progesterone production by attenuating cAMP generation and inhibiting StAR expression and P450scc activity.

4-Alkylphenols and related chemicals show similar effect on the function of human and rat estrogen receptor alpha in reporter gene assay.

Alkylphenols (APs) are widely used as important industrial materials and have attracted lots of attention because of their potential estrogenic activities. In this study, we developed human estrogen receptor alpha (hERalpha) and rat estrogen receptor alpha (rERalpha) mediated reporter gene assays and compared the estrogenic activity of APs and related chemicals based on the two ERalpha. Human breast cancer cell line MCF-7 was co-transfected with Gal4-fused hERalpha and corresponding reporter plasmid; African green monkey kidney cell line CV-1 was co-transfected with rERalpha and reporter gene. Both assays showed acceptable response to natural estrogen 17beta-estradiol (E2) with EC50 of 0.16 nM and 4.7 nM. Then the estrogenic activity of 4-APs, 4-phenylphenol and bisphenol-A were evaluated and compared with the effects of E2. The data suggested that test APs and related chemicals possessed weakly estrogenic activity and the activity of test APs increased with the increase of substituent size. This structure-activity relationship helped to infer the activity of chemicals with similar feature. Furthermore, test APs showed similar effect on the function of hERalpha and rERalpha. This consistency helped to extrapolate in vivo rodent data to human being when performing risk assessment of endocrine disruptors.

Comparative proteome analysis of neural retinas from type 2 diabetic rats by two-dimensional electrophoresis.

PURPOSE: The purpose of this study was to isolate, identify, and analyze diabetes-related protein changes that occur in neural retinas in vivo. METHODS: Total proteins were extracted from neural retinas of normal and 8-weeks diabetic Sprague-Dawley (SD) rats and separated by two-dimensional gel electrophoresis (2-DE). Some protein spots exhibiting statistically significant variations (p < 0.05) were selected randomly and identified by mass spectrometry (MS or MS/MS). The protein alphaA-crystallin was chosen as a target for specific immunodetection using Western blot to corroborate the variation found by 2-DE. RESULTS: Twenty protein spots identified included alphaA-crystallin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine (Gln) synthetase, and so forth. Western blotting analyses confirmed that alphaA-crystallin protein expression was upregulated in diabetic retina. CONCLUSIONS: In this study, we isolate, identify, and analyze diabetes-related protein changes that occur in neural retinas in vivo. Further investigation of candidate proteins may identify novel pharmacological targets for diabetic retinopathy.

[Fenvalerate affects sperm motility in SD rats].

OBJECTIVE: To observe the direct effects of fenvalerate (Fen) on sperm motility in SD rats. METHODS: Sperm were isolated from caudal epididymides of healthy adult male rats with the diffusion method. The motility parameters of the isolated sperm, such as VCL, VSL, VAP, BCF, STR and LIN, were monitored by computer-assisted sperm analysis (CASA) system after 1, 2 and 4 h Fen-exposure in vitro at concentrations of 0, 1, 4, 16 and 64 micromol/L respectively. RESULTS: After 1 and 2 h Fen-exposure, VSL, BCF, STR and LIN decreased significantly at 64 micromol/L compared with the control group. After 4 h Fen-exposure, the motility parameters VCL, VSL, BCF, STR and LIN dropped progressively at 64 micromol/L, and VCL declined markedly at 16 micromol/L. However, only VCL and STR showed alterations in a time-response manner. CONCLUSION: Fen may affect the caudal epididymal sperm and produce a direct toxic effect on sperm motility in SD rats.

Monobutyl phthalate inhibits steroidogenesis by downregulating steroidogenic acute regulatory protein expression in mouse Leydig tumor cells (MLTC-1).

Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP), display no binding affinity for the androgen receptor, yet exert antiandrogenic effects by altering steroid biosynthesis. However, the mechanisms underlying this observed effect are not known. The purpose of this study was to determine the site of MBP action on steroidogenesis in vitro using mouse Leydig tumor cells (MLTC-1). Various concentrations of MBP (0, 50, 100, 200, 400, or 800 micromol/L) were added to the medium for 24 h followed by stimulation with some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC), and pregnenolone. Data showed that MBP inhibited the increases in progesterone production induced by hCG and CT. In contrast, the levels of intracellular cAMP remained unaltered. In addition, 8-Br-cAMP-stimulated progesterone production was also suppressed by MBP. These results suggested that the site in the steroid biosynthesis pathway affected by MBP occurs downstream of PKA activation in MLTC-1 cells. Moreover, incubation with 22R-HC and pregnenolone as progesterone precursors for P-450 side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3betaHSD) respectively resulted in no marked change in progesterone production, indicating that MBP did not influence P450scc and 3betaHSD but did exert an effect on cholesterol transportation into mitochondria, the rate-limiting step. These results were supported by the downregulated StAR expression seen with MBP administration, as StAR is a key factor in this process. Data indicate that MBP interfered with steroid hormone production by affecting StAR expression in MLTC-1 cells.

Effects of terephthalic acid on rat lipid metabolism.

OBJECTIVE: To study the effect of terephthalic acid (TPA) on lipid metabolism in Sprague-Dawley (SD) rats. METHODS: Five groups of SD rats that ingested 0%, 0.04%, 0.2%, 1%, and 5% TPA, respectively, were included in a 90-day subchronic feeding study. Effects of TPA on levels of serum protein, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), total antioxidative capability (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA) were observed. Urine samples were collected and analyzed for concentration of ion. RESULTS: TPA decreased the level of serum T-AOC in a dose dependent manner. The contents of serum and bladder MDA significantly decreased in 1% and 5% TPA ingestion groups. Serum CuZn superoxide dismutase (CuZnSOD) lowered in groups of 0.2%, 1%, and 5% TPA. TPA subchronic feeding had no significant influences on serum TC, LDL or HDL, but increased serum TG, TP and ALB after administration of 0.04% and/or 0.2% TPA. Concentrations of urinary Ca2+, Mg2+, Na+, and K+ were elevated in 1% and 5% TPA groups. CONCLUSION: Antioxidative potential decreased after TPA exposure. MDA increase in serum and bladder tissues was one of the most important reactions in rats which could protect themselves against TPA impairment. The decrease of serum CuZnSOD was related to the excretion of Zn2+.

[Advances in the study of steroidogenic acute regulatory protein].

The steroidogenic acute regulatory (StAR) protein is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells. The StAR protein has a high tissue specificity, located on the mitochondrial membranes of some relative cells. It regulates the transfer of cholesterin from extracellular into intracellular and plays a dominant role in steroidogenic synthesis. Recent studies have also shown that the transcription and expression of StAR are modulated not only through the cAMP-PKA dependent pathway, but also by multiple hormones and cytokines, which contributes to the regulation of cholesterin synthesis.

Metabolism of terephthalic acid and its effects on CYP4B1 induction.

OBJECTIVE: To investgate the metabolism of terephthalic acid (TPA) in rats and its mechanism. Methods Metabolism was evaluated by incubating sodium terephthalate (NaTPA) with rat normal liver microsomes, or with microsomes pretreated by phenobarbital sodium, or with 3-methycholanthrene, or with diet control following a NADPH-generating system. The determination was performed by high performance liquid chromatography (HPLC), and the mutagenic activation was analyzed by umu tester strain Salmonella typhimurium NM2009. Expression of CYP4B1 mRNA was detected by RT-PCR. Results The amount of NaTPA (12.5-200 micromol x L(-1)) detected by HPLC did not decrease in microsomes induced by NADPH-generating system. Incubation of TPA (0.025-0.1 mmol x L(-1)) with induced or noninduced liver microsomes in an NM2009 umu response system did not show any mutagenic activation. TPA exposure increased the expression of CYP4B 1 mRNA in rat liver, kidney, and bladder. CONCLUSION: Lack of metabolism of TPA in liver and negative genotoxic data from NM2009 study are consistent with other previous short-term tests, suggesting that the carcinogenesis in TPA feeding animals is not directly interfered with TPA itself and/or its metabolites.

[Effects of mono(2-ethylhexyl) phthalate on testosterone biosynthesis in leydig cells cultured from the rat testis].

OBJECTIVE: To investigate the effects of mono(2-ethylhexyl) phthalate(MEHP), the primary metabolite of di(2-ethylhexyl) phthalate (DEHP), on testosterone biosynthesis in Leydig cells cultured from the Sprague Dawley rat testis. METHODS: Based on the primary Leydig cell culture model, MEHP exposure groups involved control (0 micromol/L), 62.5, 125, 250, 500 and 1000 micromol/L. We observed mitochondria activity with the MTT method, measured the testosterone level with RIA and determined steroidogenesis acute regulatory protein (StAR) mRNA expression with RT-PCR. RESULTS: After Leydig cells were exposed to MEHP for 24 hours, the activity of mitochondria enhanced evidently at 250 micromol/L and then declined markedly at 1000 micromol/L compared with the control group (P < 0.01). The testosterone level showed an increasing tendency in both basal and hCG-stimulated states with statistical significance at 250 and 500 micromol/L compared with the control group (P < 0.01). However, the expression of StAR mRNA appeared unchanged at 62.5, 125 or 250 micromol/L, but exhibited a decreasing tendency at 500 and 1000 micromol/L (P < 0.01). CONCLUSION: ME- HP directly affected the activity of mitochondria and testosterone biosynthesis of the Leydig cells in vitro. StAR, the regulator of cholesterol transport into mitochondria, might not be responsible for the increase of testosterone biosynthesis induced by MEHP.

[Effects of dibutyl phthalate on sertoli cells of rat testis].

OBJECTIVE: The present study was performed to examine functional and structural impairment of rat sertoli cells following dibutyl phthalate (DBP) exposure. METHODS: The 6-week-old healthy male Sprague Dawley rats were randomly divided into 4 groups with 16 animals in each group. DBP dissolved in peanut oil was administered by gavage at doses of 0, 250, 500 and 1 000 mg/kg. After 2-week DBP treatment, half of the rats were sacrificed. The rest were killed following 4-week DBP exposure. Follicle stimulating hormone (FSH) was analysed by radioimmunoassay. The relative expression levels of androgen binding protein (ABP) mRNA and inhibin (INH)alpha mRNA were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The sertoli cell ultrastructures were observed by using transmission electron microscope (TEM). RESULTS: FSH levels were increased after 4-week DBP exposure with significance at doses of 250 and 1 000 mg/kg. Sperm head count and daily sperm product were decreased significantly in 500 and 1 000 mg/kg groups. The expression levels of ABP mRNA were 0.89 +/- 0.15, 0.85 +/- 0.23, 0.54 +/- 0.17, 0.52 +/- 0.16 and 0.88 +/- 0.16, 0.61 +/- 0.12, 0.48 +/- 0.15, 0.47 +/- 0.11 for 0, 250, 500 and 1 000 mg/kg after 2- and 4-week DBP treatments respectively with significance at doses of 500 and 1 000 mg/kg (P < 0.01), while the levels of INHalpha mRNA were 0.88 +/- 0.16, 0.61 +/- 0.12, 0.48 +/- 0.15, 0.47 +/- 0.11 and 0.75 +/- 0.19, 0.56 +/- 0.16, 0.53 +/- 0.08, 0.45 +/- 0.10 with significance at all exposure groups (P < 0.01 or P < 0.05). In sertoli cells of rats exposed to 1 000 mg/kg DBP, TEM photos showed more lysosomes in cytoplasm, proliferated and expanded endoplasmic reticulum and nuclei malformation. CONCLUSIONS: Sertoli cell should be one of the major toxic targets. Impairment of spermatogenesis caused by DBP should be partly due to the suppression of ABP and INHalpha biosynthesis.

[Effect of fenvalerate on the ovarian calcium homeostasis and the serum steroid hormone].

OBJECTIVE: To observe the effects of fenvalerate (Fen) on ovarian calcium homeostasis. METHODS: hGLCs were obtained from pre-ovulatory follicles in an in vitro fertilization program, and were cultured for 72 hours. Changes in cellular [Ca(2+)]i induced by Fen in hGLCs were detected with laser scanning confocal microscopy (LSCM) by using the fluorescent Ca(2+) indicator fluo-3/AM. SD female rats were divided into four groups (control, 1/15LD(50), 1/50 LD(50) and 1/250 LD(50)) in experiment. The activity of ovarian Ca(2+)-ATPase and phosphorylase A (P-a) and the contents of calmodulin (CaM) were assessed after a 30-day Fen exposure. In addition, serum estradiol-17 beta (E(2)) and progesterone (P(0)) concentration were measured by radioimmunoassay, which the sampling rats were ensured at diestrus stage before killed according to vaginal smear. RESULTS: 20.0 and 2.0 micromol/L Fen induced the increased of [Ca(2+)]i in hGLC. This [Ca(2+)]i increase mostly resulted from Ca(2+) influx in the studied concentration. Fen had shown the inhibition effects on activity of Ca(2+)-ATPase in 1/250 LD(50) group (P < 0.001) while the activity of phosphorylase A (P-a) in treated groups had significantly enhanced than those of in control. The contents of CaM in ovaries were found to be increased in treated groups. E(2) in 1/250 LD(50) group were higher while P(0) in 1/15 LD(50) group were significantly lower (P < 0.05). CONCLUSION: Exposure to Fen interferes the serum steroid hormone concentrations partly through calcium signal pathway.